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Proteomic studies of the hid1∆ and hid3∆ mutants of
Schizosaccharomyces pombe
Abdulrahman Alasmari
To cite this version:
Abdulrahman Alasmari. Proteomic studies of the hid1∆ and hid3∆ mutants of Schizosaccharomyces pombe. Genetics. Université de Bordeaux, 2015. English. �NNT : 2015BORD0142�. �tel-01617018�
Année 2015
Thèse n°2015BORD0142
THÈSE
pour le
DOCTORAT DE L’UNIVERSITÉ de BORDEAUX
Ecole Doctorale des Sciences de la Vie et de la SantéSpécialité: Génétique
Présentée et soutenue publiquement Le 16 Septembre 2015
Abdulrahman Alasmari
Né(e) le18 Décembre 1984 à Riyadh
Proteomic studies of the
hid1Δ and hid3Δ
mutants of Schizosaccharomyces pombe
Membres du Jury
M. Moreau, Patrick Univeristé de Bordeaux Président M. Gachet, Yannicl Université Paul Sabatier rapporteur M. McFarlane, Ramsay Bangor University rapporteur M. El Ghouzzi, Vincent INSERM, Hôpital Robert Debré Examinateur M. Hooks, Mark A. Univeristé de Bordeaux Directeur de thèse
Études protéomiques des mutants
hid1Δ et
hid3Δ chez Schizosaccharmyces pombe
RÉCAPITULATIF EN FRANÇAIS
Schizosaccharomyces pombe est devenu un important système modèle pour étudier
les processus physiologiques, biochimiques et génétiques chez l'homme. Ces travaux
appartiennent à un projet sur la façon dont la fonction altérée de l'appareil de Golgi
contribue à des maladies comme le cancer ou cause des anormalités génétiques. La
protéine HID-1 de C. elegans et des humains est une protéine de l'appareil de Golgi qui
appartient à la superfamille de protéines DYMECLIN. Les animaux ont à la fois un gène
HID1 et un gène DYM. Chez les humains, une expression réduite de HID1 est
impliquée dans la prolifération de tumeurs. La perte de DYM chez les humains mène à
une déformation squelettique. S. pombe a trois gènes orthologues HID-1, mais pas de
DYM. Par contraste, de nombreux eucaryotes unicellulaires et pluricellulaires n'ont
qu'un gène DYM. Les mutants de S. pombe sans Hid1 et Hid3 étaient sensibles au
stress oxydatif et la croissance de
hid3Δ a été stoppée dans des milieux de culture
minimum standard. L'insensibilité de
hid3Δ au brefeldin A, mais sa sensibilité au
golgicide, a démontrent que Hid3 fonctionne dans le transport antérograde à travers
l'appareil de Golgi. Afin d'explorer des rapports indiquant que le renouvellement des
protéines pourrait être modifié dans
hid3Δ, j'ai entrepris une étude de protéomique a la
quantification label-free. La régulation positive de la voie de signalisation MAPK de
tension a démontré que les cellules étaient dans un état de tension dans des conditions
normales de croissance. De plus, des composants de protéine dans plusieurs voies de
signalisation étaient modifiés, pouvant affecter une large gamme de processus
cellulaires.
ENGLISH SUMMARY
Schizosaccharomyces pombe has become an important model system to study
physiological, biochemical and genetic processes in humans. This work is part of a
continuing project to study how altered Golgi function contributes to diseases, such as
cancer, or causes of genetic abnormalities. The HID-1 protein of C. elegans and
humans are peripheral membrane proteins of the Golgi apparatus and are part of the
DYMECLIN superfamily of proteins. Animals have both a HID1 and a DYM gene. In C.
elegans, HID-1 maintains normal cellular growth and in humans reduced expression of
HID1 has been implicated in tumour proliferation. Loss of DYM in humans leads to
skeletal deformation and potentially mental retardation. S. pombe has three HID-1
orthologues, but no DYM. In contrast, many unicellular and multicellular eukaryotes
have only DYM. S. pombe mutants lacking Hid1 and Hid3 were sensitive to oxidative
stress and growth of hid3
Δ was stopped in standard minimal media. Insensitivity of
hid3Δ to brefeldin A but sensitivity to golgicide A demonstrated that Hid3 operates in
anterograde protein transport through the Golgi. In order to investigate reports that
protein turnover might be altered in hid3
Δ, I undertook a proteomics study using
label-free protein quantification. Up-regulation of the MAPK stress signalling pathway
demonstrated that cells were under a state of stress under standard growth conditions.
In addition, protein components of Ras signalling, microtubule dynamics and chromatin
remodelling were altered potentially affecting a wide variety of processes from cell cycle
regulation to metabolism.
Table of Contents
Récapitulatif en Français & Mots Cléts
II
English Summary & Key Words
III
Table of Contents
IV
List of Tables and Figures
VIII
Acknowledgement
X
List of Abbreviations
XI
Récapitulatif du projet en Français
XV
I. Chapter 1:
General Introduction
1
1.1 Function of the Golgi apparatus in the cell
1
1.1.1 Golgi in S. pombe
2
1.2 The role of ubiquitylation
3
1.2.1 Ub ligases and deubiquitinating enzymes
3
1.2.2. Ubiquitylation processes in the endomembrane system
5
1.2.2.1. ER-associate degradation of proteins
5
1.2.2.2. The SREBP signalling processes of the ER and Golgi.
6
1.3 Human diseases associated with Golgi malfunction
7
1.3.1 Skeletal dysplasia
8
1.3.1.1 Malfunctioning RAB33B as a cause of DMC syndrome
9
1.3.1.2 Lack of DYMECLIN function resulting in DMC syndrome.
9
1.4 Cancer as a genetic disease
10
1.4.1 Ubiquitin and cancer
12
1.5 Environmental stress signalling
13
1.6. The HID1 protein
15
1.6.1. Human HID1
15
1.6.2 C.elegans HID-1
15
1.6.3. HID1 in S. pombe
17
1.6.4 The HID1 ortholog in S. cerevisiae, ECM30
19
1.7.1 The pombe cell cycle
20
1.8 Proteomics
21
1.9. Aims of the project and organisat
23
II. Chapter 2:
MATERIALS AND METHODS
25
2.1 Phylogenetic and computational analyses
25
2.2 Charaterization of mutant growth and stress responses
25
2.2.1 Growth of S. pombe
25
2.2.2 Growth analyses of hid mutants under stress conditions
27
2.2.3 Determination of cell number
27
2.3 Genotyping of strains of S. pombe
27
2.3.1 Isolation of the DNA from S.pombe deletions mutants
28
2.3.2 Polymerase chain reaction
28
2.4 Proteomic analyses
29
2.4.1 Growth of cells
29
2.4.2 Extraction of total cellular protein
29
2.4.3 Protein quantification and visualization
30
2.4.4 Label-free proteomic analyses
30
2.5 Immunological analyses of Hid proteins
31
2.5.1. Production of Antibodies
31
2.5.2 Immunoblotting
31
2.6 Metabolite profiling
32
2.7 General Data Analysis.
32
II. CHAPTER 3:
COMPUTATION ANALYSES OF THE DYMECLIN PROTEIN
SUPERFAMILY AND THE HID-1 ORTHOLOGUES FROM S. POMBE
33
3.1 INTRODUCTION
33
3.2 RESULTS
34
3.2.1 Global phylogenetic analysis of HIDs and DYMs
34
3.2.1.1 Selection of sequences from eukaryotic taxa for phylogenetic analysis
34
3.2.1.2 Global phylogenetic analysis
40
3.2.2 Functional characterisation of the S. pombe HIDs
43
3.2.2.1 Comparison of primary sequences to eukaryotic orthologues
43
3.2.2.2. Comparative structural properties of the HIDs from S. pombe
44
3.2.2.3. Prediction of genetic and physical interacting partners
46
3.3 DISCUSSION
47
IV. CHAPTER 4:
EFFECT OF STRESS TREATMENTS ON THE GROWTH OF
HIDΔ MUTANTS
50
4.1 INTRODUCTION
50
4.2 RESULTS
50
4.2.1. Effects of stress treatments on growth of cells lacking Hids
52
4.2.2. hid3Δ mutants are resistant to Brefeldin A and sensitive to Golgicide A
54
4.3 DISCUSSION
55
V. CHAPTER 5:
PROTEOMIC AND METABOLOMICS CHARACTERIZATION OF
THE HID1∆ AND HID3∆ MUTANTS OF S. POMBE
58
5.1 INTRODUCTION
58
5.2 RESULTS
60
5.2.1. Strategy for sample preparation for metabolite and protein profiling
62
5.2.2. Metabolic consequences of removing Hid1 and Hid3
62
5.2.3 Proteomic study on the effects of missing Hid proteins
63
5.2.4. Preliminary analyses of immune-detection of Hid proteins in Pombe.
71
5.3 DISCUSSION
71
CHAPTER 6 :
GENERAL DISCUSSION AND FUTURE WORK
75
I CHAPTER 3 CONCLUSIONS
75
III CHAPTER 5 CONCLUSIONS
79
IV FUTURE WORK
83
List of Tables and Figure Chapter 1: Introduction
Figure 1.1. Trafficking through the endomembrane system in yeast.
Figure 1.2 Diagram of the Golgi apparatus and ER exit sites in yeast species. Figure 1.3. The alternative growth stages of C. elegans.
Figure 1.4 Schematic diagram of the possible role of S. pombe Ftp105/Hid3-mediated protein trafficking from the Golgi.
Chapter 2 Materials and Methods
Table 2.1 Chemical Stress agents.
Table 2.2 List of genotypes used for growth analyses
Chapter 3 Computation analyses of the DYMECLIN protein superfamily and the HID-1 orthologues from S. pombe.
Table 3.1. List of evolutionary lineages containing putative DYMs or HIDs.
Table 3.2. Quantitative comparison of primary sequences between members of the HID and DYM superfamily.
Table 3.3. GO ontology references to potential function of Hid proteins from S. pombe. Figure 3.1. Presence of HID (A) or DYM (B) orthologues in the higher phylogenetic groupings. Figure 3.2. Phylogenetic groupings of the HIDs and DYMs.
Figure 3.3. Multiple sequence alignment comparing S. pombe Hids with HID1 from C. elegans and H.
sapiens.
Figure 3.4. Comparison of HID domain structures.
Figure 3.5. STRING output for pombe and human HID interactions. Figure 3.6. Model of Hid3 function.
Chapter 4 Effect of stress treatments on the growth of hidΔ mutants
Table 4.1. A summary of the effect of general stresses on the growth of mutant genotypes. Table 4.2. A summary of the effect of DNA damaging agents on growth of mutant genotypes. Figure 4.1. Relative expression of hid genes under stress.
Figure 4.2 Effects of oxidative stress on mutant growth. Figure 4.3. Differential growth of mutants on minimal medium.
Figure 4.4. The effect brefeldin A (BFA) on mutant growth. Figure 4.5. The effect of Golgicide A (GA) on mutant growth.
Figure 4.6. Hid3 involvement in protein transport through the Golgi Apparatus.
Chapter 5 Proteomic and metabolomics characterization of the hid1∆ and hid3∆ mutants of S.
pombe
Table 5.1. Scheme of preparation of samples for post-genomic studies. Table 5.2. Metabolite absolute values as determined by 1H-NMR. Table 5.3. Summary statistics for proteomic analyses.
Table 5.4. List of proteins in hid1∆ with significantly altered amounts. Table 5.5. Proteins significantly altered in abundance in hid3∆.
Figure 5.1. Global comparison of metabolite data among mutant and WT genotypes. Figure 5.2. Denaturing PAGE of protein extracts used for proteome analyses. Figure 5.3. Denaturing PAGE preparation of samples for trypsin digest. Figure 5.4. PCA of normalised values of quantified proteins.
Figure 5.5. The numbers of proteins significantly altered in amount.
Figure 5.6. Ontology analysis of proteins with significantly altered amounts. Figure 5.7. Immuno-detection of Hid proteins.
Acknowledgments
This work was funded by Tabuk University in Saudi Arabia. I extend my fondest thanks to my family, who supported me during my PhD journey. My deep gratitude also goes to my supervisor, Dr. Mark Hooks, for all his guidance, support and encouragement. Many thanks are extended to all the people who supplied me with strains, especially Dr. David Pryce. I also would like to thank Dr. Marc Bonneu and Dr. Stephane Claverol of the Proteomics Platform at the Centre Génomique Fonctionelle de Bordeaux for conduction the protein quantification proteomics data. I also gratefully acknowledge the support from all my colleagues, past and present, and especially the members of the INRA Labs.
Abbreviations
1-D: one-dimensional gel electrophoresis
1H NMR : H-NMRhydrogen-1 NMR
AtDYM: DYM from Arabidopsis ATM: ataxia telangiectasia mutated Aβ: amyloid-beta
BFA: Brefeldin A Bp: base pairs
BSA Bovine Serum Albumin C.elegance: Caenorhabditis elegans
c10orf76: chromosome 10 open reading frame 76 c17orf28: chromosome 17 open reading frame 28 CDKs: catalytic cyclin-dependent kinases CeDYM: DYM from C. elegans
CESR: core environmental stress response CMM: Cisternal Maturation Model
CPT: camptothecin
CSDE1: cold-shock domain-containing protein
Da: Dalton
Daf-c: Dauer formation consistitutive Daf-d : Dauer formation defective
DMC1: DOWNREGULATED IN MULTIPLE CANCERS 1 DNA: deoxyribonucleic acid
DNA-PK: DNA-dependent protein kinase DR: down-regulated or less abundant DUB: deubiquitinating enzyme
DYM: Dyggve-Melchior-Clausen syndrome (DYMECLIN) e.g. for example
EDTA: Ethylenediaminetetraacetic acid solution EMM: Edinburgh Minimal Medium
ER: Endoplasmic Reticulum
ERES. Endoplasmic reticulum exit sites ERK: extracellular-signal regulated kinase G418: Geneticin
GCA: Golgicide A GE: Generation Time
GEFs: guanine nucleotide exchange factors GO analysis: Gene Ontology Analysis
GRASPs: Golgi reassembly stacking proteins HID-1: Heat-Induced Dauer formation protein 1 HIS: histidine
HR: homologous recombination HsDYM: DYM from humans
HSPs: heat shock proteins HU: hydroxyurea
i.e. that is
ITRAQ: Isobaric tag for relative and absolute quantitation JAMM : JAB1/MPN/Mov34 metalloenzyme domain proteases KCl: Potassium chloride
L1-L4: four larval stages
LC-MS/MS: liquid chromatography-tandem mass spectrometry LOH: Loss of Heterozygosity
Lycopsida: lycophyte m/z: mass to charge
MALDI-ToF MS: ionization time of flight mass spectrometry MAPK: Mitogen-activated protein kinase
Min: minutes
MJD : Machado-Josephin domain proteases MMS: methyl methanesulphonate
mRNA: Messenger RNA
MS/MS: tandem mass spectrometry MS: mass spectrometry
MTC: mitomycin C Multi-Ub: multi ubiquitin
NCBI: National Center for Biotechnology Information NMR: nuclear magnetic resonance
ORFs: open reading frames OTU : ovarian tumour proteases
PAGE: polyacrylamide gel electrophoresis PCA : Principal Component Analysis PCR: Polymerase Chain Reaction PI3K: phosphoinositol-3-Kase Poly-Ub: poly ubiquitin
P. pastoris: Pichia pastoris
PVDF: Polyvinylidene fluoride membrane r.p.m.: revolutions per minute
RNA: ribonucleic acid
ROS: reactive oxygen species S. cerevisiae: Saccharomyces cerevisiae S. pombe: Schizosaccharomyces pombe
SAPK1: stress activated protein kinase pathway 1 SDS: sodium dodecyl sulfate
SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis Sec: seconds
SESR: stress-specific environmental stress response SMC: McCourt dysplasia
SREBP: Sterol Receptor Element Binding Protein SUMO: small Ub-like modifier
TBZ: thiabendazole TGN: trans-Golgi network TOF: time of flight
TSGs: Tumour Suppressor Genes Ub: ubiquitin
UBA: ubiquitin associated domain UBL: ubiquitin-like domain
Ubp: ubiquitin specific protease UCH : ubiquitin C-terminal hydrolases UR: Up-regulated
URA: uracil
USPs : ubiquitin-specific proteases UV: ultra violet
URA: uracil
USPs : ubiquitin-specific proteases UV: ultra violet
VCN: Vector controls NatR WT: wild type
YE: yeast extract
Récapitulatif du projet en Français
Introduction
Les protéines destinées à être sécrétées ou transférées vers un compartiment particulier sont fabriquées dans le réticulum endoplasmique (RE), puis elles doivent être traitées pour les rendre entièrement fonctionnelles. C'est une des principales fonctions de l'appareil de Golgi. L'appareil de Golgi est divisé en compartiments appelés citernes. Les protéines issues du RE pénètrent dans l'appareil de Golgi par la face-cis et ressortent par la face-trans. Il en résulte que toutes les protéines doivent passer dans la région dite de citerne, qui contient les enzymes divers, appelés enzymes résidents, pour faire les modifications comme la glycosylation, la sulfatation ou la phosphorylisation.
L'Ubiquitine (Ub) est une petite protéine (76 aminoacides), compacte et superpliée, qui est fortement conservée dans les organismes eucaryotes. Il existe trois différences d'acides aminés entre la levure et l'homme (Shaid et al., 2013). Les cellules utilisent Ub comme une étiquette pour modifier le contenu des protéines en fonction des conditions cellulaires pendant la dégradation des protéines. La voie Ub est aussi une modification post-translationnelle des protéines qui est importante et étendue, mais ne se limite pas à la dégradation. L'ubiquitylation guide la fonction protéique dans la réparation de l'ADN, le transfert protéique, la modification de la structure protéique, la modulation de l'activité des protéines cibles, la croissance cellulaire et la transduction de signal (Horák, 2003). L'ubiquitination est un processus important dans le système de la membrane endosomale qui comprend le réticulum endoplasmique et l'appareil de Golgi. L'ubiquitylation est importante et permet aux cellules de vérifier que seules les protéines fonctionnelles sont modifiées et finalement sécrétées. La plupart des protéines qui sont ubiquitinées dans une cellule sont des protéines nouvellement synthétisées qui sortent juste de ribosomes (Kim et al., 2011) et il est estimé que l'ubiquitylation pourrait servir de système de contrôle de la qualité pour les protéines nouvellement synthétisées (Chhangani et al., 2012). Cela constitue aussi une partie d'un processus important de signalisation pour permettre à l'appareil de Golgi de communiquer avec le noyau. Par exemple, le contrôle des niveaux de cholestérol dans les cellules humaines passe par la voie de la protéine se liant à l'élément récepteur du stérol (SREBP). Autant chez l'homme que S. pombe, le clivage de l'extrémité N-terminale de SREBP dans la membrane de l'appareil de Golgi est initié par son ubiquitylation (Stewart et al., 2011). Au cours des dernières années, un grand nombre de maladies a été lié
au mauvais fonctionnement de l'appareil de Golgi, à l'inclusion de maladies cutanées, de troubles neurologiques, de dysplasies squelettiques et de troubles des tissus conjonctifs (Bexiga and Simpson, 2013).
Le gène de la protéine 1 de formation de larves dauer induite par des températures
élevées (hid-1) de C. élégans a été trouvé dans un dépistage à la recherche de mutants du
nématode, qui a adopté la formation de larves Dauer dans de bonnes conditions de croissance, mais avec une température légèrement plus élevée (Ailion and Thomas, 2003). HID-1 est une protéine de la membrane de l'appareil de Golgi qui semblerait être impliquée dans l'excrétion de neuropeptides dans C. elegans. Il a été montré que le gène orthologue S. pombe de HID-1 localise le déubiquitinase Ubp5 (gène orthologue S. pombe de USP7 chez l'homme) dans l'appareil de Golgi. La translation fonctionnelle chez l'homme est qu'un mauvais fonctionnement de la protéine HID1 ou son absence affectera la place dans la sous-cellule de l'enzyme déubiquitylatant USP7.
Objectifs du projet et organisation de la thèse
Le but de l'introduction fournie dans le chapitre 1 était de présenter l'idée que la modification de la fonction de l'appareil de Golgi a des effets au delà du transport vésiculaire. Ceci est intimement lié au fonctionnement général des cellules à travers un certain nombre de systèmes de signalisation. L'ubiquitinylation était au centre de cette introduction parce que la manipulation des processus Ub est une des fonctions proposées de la protéine Ftp105/Hid3 de
S. pombe. Toutefois, il est probable que la protéine Ftp105/Hid3 ait d'autres fonctions dans
l'appareil de Golgi ou qu'elle puisse affecter d'autres processus quand elle est mutante. La question demeure aussi concernant le rôle des deux gènes paralogues Ftp105/Hid3. Les travaux du groupe, dont ce projet de thèse fait partie, utilisent des technologies postgénomiques de transcriptomique, protéomique et métabolomique pour avoir une meilleure idée de ce que font ces protéines, pour étudier les réactions des mutants à la tension sans remplacer les gènes
hid, puis pour déterminer les conséquences sur le métabolome et le protéome des mutants. Les
données de ces travaux sont présentées dans trois parties principales (chapitres 3 à 5) pour finir par une discussion générale sur le projet et une proposition d'orientations futures des travaux (chapitre 6). Les résultats sont résumés pour chacune de ces parties. Le chapitre 2 détaille la méthodologie et n'a donc pas été inclus.
Chapitre 3: Analyses de calcul de la superfamille de protéines DYMECLIN et les gènes orthologues HID-1 issus de S. pombe
Le chapitre 3 décrit les travaux de calcul pour analyser les HID et leurs relations avec leur membre le plus proche, le DYMECLIN (DYM). Les travaux phylogénétiques ont été conduits pour déterminer s'il pouvait être prouvé que des fonctions des deux protéines se chevauchaient. La présence ou l'absence de HID et DYM a été vérifiée dans les supergroupes et les taxons de premier rang de NCBI (tableau 3.1). Les animaux présentaient des protéines HID et DYM, leur lieu et leur action, comme l'association dynamique avec l'appareil de Golgi, semblaient être très similaires. Il est évident que les eucaryotes peuvent avoir des protéines DYM et/ou HID tandis que les animaux ont les deux. Les champignons, sauf quelques exceptions rares, n'ont que des protéines HID et les plantes n'ont que des DYM (figure 3.1, 3.2). La phylogénie et les comparaisons de séquences ont confirmé la présence de trois gènes orthologues HID1 dans S. pombe et présentaient une séquence de diversification (figure 3.3, tableau 3.2). J'ai proposé que Ftp105 soit appelé Hid3 à l'avenir. S. pombe représente un bon modèle pour étudier la fonction de Hid, parce que cela élimine les complications potentielles de la présence de protéines DYM, mais il est possible qu'il y ait une fonction redondante au sein des SpHid.
Des informations de bases de données disponibles au public indiquent que Hid1 et Hid2 se trouvent dans le cytosol/noyau avec une fonction inconnue, tandis que Hid3 est une protéine de l'appareil de Golgi (mais aussi peut-être cytosolique) et responsable de la position des protéines dans l'appareil de Golgi (tableau 3.3). Une analyse structurelle des données de séquence suggèrent que SpHid1 est plus étroitement liée aux HID animaux, mais il semble que SpHid3 a conservé les propriétés structurelles qui lui permettent de se fixer à l'appareil de Golgi (figure 3.4). Enfin, des données disponibles au public ont été examinées pour découvrir des associations possibles de chacun des Hid avec d'autres protéines qui pourraient révéler des aspects de fonction (figure 3.5). Les Hid n'ont présenté qu'un nombre très faible d'associations, mais il a été démontré que Hid3 était associé à Ubp5 comme des rapports l'ont indiqué. De cette association, un modèle de base a été créé pour montrer comment la modification des niveaux de protéine Hid3 peut affecter les niveaux d'autres protéines à travers Ubp5 et au final le métabolisme et l'expression génique (figure 3.6).
Chapitre 4 : Effet des traitements de la tension sur la croissance de mutants hidΔ Des données déjà publiées suggéraient que hid1+ et hid2+ peuvent être impliquées
dans les réactions à la tension, parce qu'elles sont induites en réaction à une tension (figure 4.1). Il a été rapporté que le mutant hid3 est sensible à certains traitements, comme des doses de sel élevées et certains produits pharmaceutiques (www.pombase.org). Les mutants produits dans l'étude d'Alshehri (2015) ont donc été testés pour déterminer leurs réactions de croissance à des tensions/traitements chimiques divers (tableaux 4.1, 4.2). Aucun des mutants simples
hid1Δ, hid2Δ et hid3Δ n'était sensible à une gamme variée de tensions, sauf au stress oxydatif,
qui affectait hid1Δ et hid3Δ en ralentissant fortement la croissance des cultures (figure 4.2). Il est intéressant de noter que la croissance de hid3Δ a été sérieusement affectée quand elle était cultivée dans des milieux de culture minimum (figure 4.3). On n'en connaît pas la cause, mais je spécule que hid3Δ manque d'un nutriment essentiel. Pour finir, en utilisant les inhibiteurs de l'appareil de Golgi brefeldin A (figure 4.4) et golgicide A (figure 4.5), j'ai pu déterminer que dans
S. pombe, Hid3 fonctionne dans le transport antérograde des protéines (figure 4.6).
Chapitre 5 : Caractérisation métabolomique et protéomique des mutants hid1Δ et hid3Δ de S. pombe
Ce chapitre décrit les propriétés métabolomiques et protéomiques des mutants. La métabolomique sert à juger dans quelle mesure la physiologie et la biochimie fondamentales des cellules se modifient quand une mutation génétique est introduite. Par ailleurs, les évolutions métaboliques peuvent fréquemment expliquer pourquoi la croissance cellulaire est modifiée. Les échantillons utilisés dans l'étude étaient exactement les mêmes que ceux utilisés dans l'étude transcriptomique (tableau 5.1). Un nombre limité de métabolites cellulaires a été quantitativement mesuré avec 1H-NMR. Ces mesures étaient particulièrement remarquables en raison de l'augmentation de la quantité de glycérol et de tréhalose présents dans hid3Δ. Ces composés sont synthétisés en réaction à la tension, mais ils sont aussi produits en adaptant les cellules à un état quiescent. Une analyse en composantes principales des données du métabolite a montré que l'état métabolique de hid1Δ et de hid3Δ étaient différents de la variation contingente négative (VCN) de contrôle (figure 5.1).
La conduite d'une étude protéomique visait un but directement lié à l'implication potentielle de Hid3 dans les processus utilisant l'ubiquitine qui impliqueraient la dégradation des protéines. Il est clair que le champ de la protéomique est large et qu'il peut y avoir plusieurs façons dont l'affectation de l'appareil de Golgi peut modifier les niveaux protéiques, mais la première étape était de déterminer les protéines qui évoluaient et de combien. Des protéines totales ont été extraites des mêmes échantillons utilisés pour l'étude métabolomique. Par la
suite, une électrophorèse SDS-PAGE a montré que les génotypes ne présentaient pas de grandes différences au niveau de l'abondance des protéines, puisque les schémas présentés par les bandes et les quantités de protéines apparaissaient uniformes (Figure 5.2). Les échantillons de protéine ont été envoyés à l'unité de protéomique de CGFB où 10 μg de la protéine totale de chaque échantillon a été purifiée par gel et digérée dans le gel avec de la trypsine (figure 5.3). Les protéines ont été quantifiées par une analyse du mélange résultant de peptides au moyen de la spectrométrie de masse (tableau 5.3). L'analyse en composantes principales (figure 5.4) et la quantification de protéines individuelles (Figure 5.5) a montré que la composition de la protéine hid3Δ était fortement différente de celle d'autres génotypes. L'analyse ontologique des protéines en évolution a montré pour hid3Δ des niveaux élevés de protéines de réaction à la tension et des niveaux réduits de protéines biosynthétiques (figure 5.6), indiquant des cellules dans des conditions chroniques de tension. L'inspection de protéines individuelles qui ont été modifiées en abondance a révélé la régulation positive dans
hid3Δ de points clés de la voie de signalisation MAPK de réaction à la tension (Tableau 5.5).
D'autres protéines intéressantes faisant l'objet d'une augmentation étaient les protéines de remodelage de la chromatine Obr1 et l'histone H2B qui affecteraient l'expression génique et la GTPase Ras1, qui est une protéine clé de voie de signalisation dont la fonction dépend de sa place au sein de la cellule. Ces différences montrent que l'élimination de Hid3 engendre un éventail large d'effets sur la fonction cellulaire.
Chapitre 6 : Discussion générale et travaux futurs
Ce chapitre présentait les conclusions obtenues par chaque partie de l'étude. Les principales conclusions du chapitre 3 étaient la confirmation de plusieurs gènes orthologues HID1 dans S. pombe et l'absence de DYM. En conséquent, S. pombe est un modèle qui convient dans l'étude de la fonction de Hid. Du fait de leur absence de HID, d'autres organismes comme les plantes plus grosses pourraient être utilisés pour étudier la fonction de DYM. L'étude phylogénétique n'a pas abouti à la fondation de l'origine de la superfamille de DYM, mais il est clair que ces protéines sont apparues avec la diversification majeure des organismes eucaryotes. La caractérisation phylogénétique de la superfamille de DYM demande des travaux plus importants. Les principales conclusions des tests de tension présentées au chapitre 4 étaient la sensibilité de hid1Δ et hid3Δ aux traitements oxydants, l'absence de croissance de
hid3Δ dans des milieux de culture minimum et la confirmation du fonctionnement de Hid3 dans
traitement à l'eau oxygénée pure et pourquoi le mutant hid2Δ pouvait inverser l'effet. Il est probable que hid3Δ subissait déjà des tensions et que des réactions étaient déjà activées de sorte qu'il ne pouvait pas gérer l'insulte supplémentaire du traitement à l'eau oxygénée pure. L'incapacité de hid3Δ à se développer dans des milieux de culture minimum suggère qu'il lui manque un nutriment essentiel qui peut être acquis par des souches non affectées. Ceci va dans le même sens que l'induction de l'expression des gènes encodant les protéines de transport (Alshehri 2015). Le chapitre 5 a confirmé autant pour le métabolisme que les niveaux de protéines que les cellules de hid3Δ subissent une forte tension même dans des conditions normales de croissance. Les évolutions des niveaux de métabolite et de protéine sont cohérentes avec l'idée que la croissance réduite des cellules de hid3Δ peuvent être dans un état de quiescence partielle et les évolutions des niveaux de protéine présentent le mécanisme qui explique les modifications observées dans l'expression génique (figure 6.1). Toutefois la gamme large de protéines dont les niveaux ont été modifiés montre que de nombreux processus différents seront affectés dans les souches de hid3Δ. Enfin, je discute de plusieurs voies possibles de recherche allant de l'utilisation des HID et DYM humains et de C. elegans pour inverser les phénotypes hid3Δ jusqu'à la caractérisation biochimique et structurelle des associations de Hid3 avec la membrane de l'appareil de Golgi. Il faudra déterminer l'emplacement dans les sous-cellules des protéines de signalisation, comme Ras1 ainsi que les mécanismes qui servent à modifier les protéines soit par l'augmentation de l'expression des gènes, soit par la modification de la dégradation.
Figure 1.1. Trafficking through the endomembrane system in yeast. Proteins synthesized in the
rough endoplasmic reticulum (ER) are passed to the Golgi apparatus by COPII protein complex coated vesicles in endoplasmic reticulum (ER)-Golgi intermediate compartment (ERGIC). As the proteins and lipids traverse the Golgi, they are processed and/or modified and subsequently sorted to various compartments within the cell, such as the vacuole of plasma membrane, or secreted outside of the cell. The image was taken from Aguiar et al., (2014).
Chapter 1
General Introduction
1.1 Function of the Golgi apparatus in the cell
There are two main types of cells, prokaryotic and eukaryotic, which form the basis for cellular life. They carry the basic things that all cells require, such as a cell membrane, DNA, cytoplasm, and ribosomes (Alberts et al., 2002). Eukaryotic cells differ from prokaryotic cells in that they can be unicellular like prokaryotes but have a diverse range of cell shapes and sizes and can combine to form multicellular organisms. On the cellular level there are fundamental differences between eukaryotic and prokaryotic cells, such as a nucleus, and organelles, such as mitochondria or plastids, that can generate energy. Eukaryotic cells also have a well-defined and intricate endomembrane system that transports proteins to different parts of the cells and allows cell to cell communication in multicellular eukaryotes. The group is interested in a part of this endomembrane system known as the Golgi apparatus, which is responsible for the processing and packaging for sorting proteins and lipids to other parts of the cell, such as the lysosome in animal cells or the vacuole in plants and fungi, or to the outside of the cell (Glick, 2000). The Golgi is an organelle that consists of various numbers of membrane stacks that various in number and shape depending on the organism. The Golgi apparatus is comprised of four main parts, cis-Golgi, medial-, trans-Golgi and trans-Golgi network (TGN). The terms ‘cis’ and ‘trans’ refer to the different faces of a Golgi complex. Vesicles arrive at the cis-face from the Endoplasmic Reticulum (ER) and they leave from the trans-face to travel to the plasma membrane or an interior compartment (Figure. 1.1) (Nakano and Luini, 2010).
Proteins are found everywhere in the cell as they are the ‘workers’ of the cell providing structure and catalysing reactions. Protein function is so closely related that when a protein does not function properly it can have bad effects on a cell leading to different types of congenital diseases, such as muscular dystrophy, Alzheimer’s disease and cystic fibrosis. Most importantly for my study is the group of proteins that can be directed to specific places in the cell, for instance the plasma membrane, the cytoplasm, the nucleus, the endosomes/vacuole, and the cellular machinery that accomplishes this, namely the Golgi apparatus. The diseases mentioned above relate to disrupted protein trafficking by Golgi apparatus (Ungar, 2009).
Proteins destined for secretion or for transfer to a particular compartment are made in the ER and then they need to be processed to make them fully functional. That is one of the
main functions of the Golgi apparatus. The Golgi is divided into compartments called cisternae. Proteins enter the Golgi from the ER by cis-face and they exit from trans-face side.
As a result, all proteins have to pass a region called cisterna which contains the various enzymes, called resident enzymes, for doing protein modifications, such as glycosylation, sulfation, or phosphorylation. There are two theories about how proteins move through the Golgi, the Cisternal Maturation Model (CMM) theory and the Vesicular Model (VM) theory. In the CMM, the Golgi cisternae themselves mature and convert to the next type and in doing so carry their cargo from cis-face to trans-face. The VM model states that proteins are passed to fixed cisternae through vesiculation (Connerly, 2010). Using GFP-labelled proteins in budding yeast, two independent laboratories provided evidence for the CMM model to show that resident proteins move through the stacks (Losev et al., 2006; Matsuura-Tokita et al., 2006), so this remains the preferred theory. However, both types of movement are likely to occur, because proteins can be recycled back through the Golgi, which is likely mediated by vesicle transfer (Emr et al., 2009).
1.1.1 Golgi in S. pombe
There are a number of different Golgi structures that have been identified in various organisms, but the typical structure as cis-, medial-, and trans-cisternae plus the trans-Golgi network (TGN). These compartments have been found to differ from each other in properties, like cytochemical-staining, composition of resident enzymes, and ability to bud COPI- or clathrin-coated vesicles (Farquhar and Palade, 1981; Kleene and Berger, 1993; Nilsson et al., 2009; Rabouille et al., 1995; Staehelin and Kang, 2008). Resident Golgi proteins can move rapidly within and between compartments. For example, according to the cisternal maturation model mentioned above, glycosylation enzymes that operate in assembly line fashion to process secretory cargoes must be re-transported by to the original site of function if not degraded (Glick and Luini, 2011; Nilsson et al., 2009; Rabouille et al., 1995). A study by Glick and Luini, (2011) tried to investigate various models known to be linked to Golgi traffic, such as vesicular transport between compartments and cisternal maturation, to provide a unifying observation for Golgi function. They concluded that no single model can easily explain all of the observations from diverse organisms. A study by Klute et al., (2011) compared the origin and history of the Golgi by comparing proteins involved in Golgi morphology as well as those involved in trafficking, by choosing proteins that act as carriers to and from the Golgi at both the
cis- and trans-faces. They concluded that the basic elements of vesicle trafficking and Golgi
Figure (1.2). Diagram of the Golgi apparatus and ER exit sites in yeast species. (A) Ordered Golgi
stacks locate close to the ERES (light blue) in P. pastoris similar to S. pombe. The cis cisterna (red) of Golgi stack faces ERES. (B) in S. cerevisiae, Golgi cisternae are it matures into a medial and a trans cisterna and dispersed to the cytoplasm (Suda and Nakano, 2012).
Many of the processes of the Golgi complex of higher eukaryotes like mammals, such as carbohydrate modification and proteolytic processing, have been found in to occur in the yeast Golgi (Seemann et al., 2000).There would not be expected to be identical functions of the Golgi between higher eukaryotes and the yeast, because the Golgi in higher eukaryotes is necessary for processing and secreting many of the factors that are necessary growth, proliferation and cell communication within a multicellular context. The internal cell structure of mammalian cells is also different to that of yeast, where mammalian cells have an endosomal system for degrading cellular components, where yeasts have a vacuole system. The structure of the Golgi apparatus among yeast is also different where yeasts like S. pombe (Chappell and Warren, 1989) and P. pastoris (Preuss et al., 1992; Rossanese et al., 2001) have a stacked lamellae structure like higher eukaryotes, but S. cerevisiae has a more dispersed type of Golgi structure lacking visible cisternae (Figure 1.2). However, Mammalian-like Golgi reassembly stacking proteins (GRASPs) have been discovered in the three yeast species noted as factors involved in the formation of Golgi stacks (Vinke et al., 2011). Moreover, in yeast and unlike the mammalian cells, the Golgi down during the cell division to partition during cytokinesis (Preuss et al., 1992; Rossanese et al., 2001).
However, even in a dispersed state, the Golgi must be functional in S. pombe in order to form the septum. More investigations are needed to identify the similarities and differences in the nature of the Golgi complex, because the Golgi is an ancient and ubiquitous feature of eukaryotic cells. In addition, the use of various yeast species will help us to understand the importance of Golgi stack formation.
In addition to the morphology of the Golgi, there are functional differences among the yeasts as revealed in differences in protein components. For example, Rho3 has distinct roles in
S. pombe and S. cerevisiae. In S. cerevisiae it appears to be involved in influencing cell growth
by regulating polarized secretion through the actin cytoskeleton by interacting with Exo70 and Myo2 (Robinson et al., 1999). In S. pombe, Rho3 plays an important role in Golgi/endosome trafficking by complexing with Apm1 and other subunits of the AP-1 complex. Another main feature differing S. pombe from S. cerevisiae and other eukaryotes is that it that has only one Ras protein. There are two Ras proteins in S cerevisiae and four in mammals. This S. pombe Ras displays differing localisation (Fukui et al., 1986; Nielsen et al., 1992), which is known to influence the pathways it controls, such as the byr2/byr1/spk1 mitogen- activated protein kinase cascade. It also activates the the Rho GTPase Cdc42 protein (García et al., 2006) pathway to regulate cell polarity by modulating cytoskeletal elements and maintaining elongated cell morphology and protein trafficking. The differential location of Ras raises important questions as
to how do signalling proteins selectively regulate particular pathways. Mammalian and S. pombe Ras proteins have significant sequence homology that makes S. pombe an ideal organism to study aspects of Ras signalling, as it contains only a single Ras protein.
1.2. The role of ubiquitylation
Ubiquitin (Ub) is a small (76 amino acids), compact and super-folded protein which is highly conserved in eukaryotic organisms. Between yeast and humans there are only three amino acid differences (Shaid et al., 2013). Cells use Ub as a tag to change according to cellular conditions. The cell must respond to changing conditions by producing the proteins it requires at the moment. Accordingly, it is beneficial to the cell to degrade or recycle those proteins that are not required. Ubiquitylation has evolved to be a principal mechanism where proteins are tagged by direct binding with Ub to tag them for degradation by the proteasome (Hershko and Ciechanover, 1998). The Ub/proteasome pathway also is important to keep the cells healthy and functioning in presence of translation errors and genetic mutations that damage proteins (Tu et al., 2012). Misfolded proteins can be rescued by repair proteins called chaperones, but if not, the protein will be directed to degradation by the Ub/proteasome pathway. The Ub pathway is an important and widespread post-translational modification of proteins, but not only for degradation. Ubiquitylation guides protein function in DNA repair, protein translocation, altering protein structure, modulating activity of the target proteins, cell growth and signal transduction (Horák, 2003).
There are also different kinds of Ub-like proteins (UBLs), such as SUMO (small Ub-like modifier), which have some similarities in structure with Ub. In humans, there are 10 genes encoding UBLs called NEDD1 – NEDD10. Like Ub, NEDD UBLs are conserved among eukaryotes. They also have the ability to attach the target proteins to modify their function or stability, and have an important role in cell cycle control and embryogenesis (Brown and Jackson, 2015). The process of NEDD-UBL ligation is known as neddylation and it shows similarity to ubiquitylation in that NEDDs must be processed before attachment to protein substrates.
1.2.1 Ub ligases and deubiquitinating enzymes
Ub is added covalently to a target lysine residue on a protein target either as a single protein, as a chain of Ub proteins (poly-Ub) and/or on multiple sites on the same protein (multi-
Ub). Poly-Ub is the process for tagging proteins for degradation, whereas mono-Ub generally affect protein localization and multi-Ub can affect protein function (Xu and Jaffrey, 2011). The process of ubiquitylation is a three-step process performed by what are called Ub ligases. E1 ligase is the first step and activates Ub for attachment to Ub E2 ligase, which is also called ubiquitin conjugating enzyme. Thirdly, Ub is transferred from E2 onto the C-terminus of E3, which then attaches Ub on to specific lysine residues of target proteins by ac C-terminal glycine. E3 Ub ligases are the most abundant of the three types, because they must recognise distinct molecular targets. There are more than 600 putative E3 ligases in the human genome (Li et al., 2008a). There are two main families of E3 ligase, the HECT family and the RING E3 ligases. The HECT family of E3 ligases transfer Ub to a target protein. The RING E3 ligases probably serve as recognition helpers for E2 to transfer Ub directly to the target (Weissman, 2001).
If the classical function of Ub is to tag proteins for degradation, it means the function of deubiquitinating enzymes (DUBs) is likely to be protein stability. Thus, based on the on the type of Ub chain that is attached to the substrate, deubiquitinating enzymes can start to react with target proteins. There are three general roles for DUBs. First, DUBs are important for Ub maturation once synthesised, since they can be synthesized in poly Ub chains or attached to smaller peptides. Secondly, DUBs can reverse the ubiquitylation action that targets proteins for degradation and in the process maintain Ub homeostasis through Ub recycling. Thirdly, DUBs can alter the type of Ub modification of a target enzyme (Komander et al., 2009). The human genome contains about 90 DUBs, but with only about 80 considered to be active (Clague et al., 2012). These proteins have been classed into 5 families depending on the motif architecture, UCH, OTU, MJD JAMM, and USPs. The USPs comprise the largest family DUBs with 63 members, and this family is considered to be responsible for cleaving Ub to prevent protein degradation by the proteasome. USPs have an important role to counteract the Ub ligases. In S.
pombe there are 20 DUBs, of which Ubp5 is of interest to us due to its interaction with Hid3
(Kouranti et al., 2010a).
1.2.2 Ubiquitylation processes in the endomembrane system 1.2.2.1 ER-associate degradation of proteins
It is interesting that most of the proteins that are ubiquitinated in a cell are newly synthesized proteins just coming off of ribosomes (Kim et al., 2011). It is thought that ubiquitylation may serve as a quality control check system for newly synthesized proteins (Chhangani et al., 2012). Within the ER there is also a quality control system that makes sure that only properly-folded, functionally-active membrane proteins are transported onto the Golgi.
It is thought that glycosylation of proteins and insertion in to the membrane makes them much more resistant to deactivation and degradation so a screening system is required to remove unfunctional proteins. This is called the ERAD system (Kornitzer and Ciechanover, 2000) and associated with the Unfolded Protein Response. In humans, unproperly folded proteins appear to be recognized by molecular chaperones, such as the heat shock proteins (HSPs) and marked by phosphorylation by three particular membrane associated kinases PERK, IRE1, and ATF6. This slows down protein translation at the ER and permits targeting of unfolded proteins by an unknown E3 Ub ligase (Buchberger et al., 2010). Ubiquitylation is the signal that the protein is to be removed from the ER membrane and sent to the proteasome for degradation. In order to help decide between which proteins are to sent for degradation and those that are kept is discrimination made by the E3 ligase, but fine-tuned by the ER resident DUBs (Zhang et al., 2013b). Except for certain components, for example the chaperones and E3 ligases used, the ERAD system is conserved in the yeasts and the study of these systems has provided much insight into the animal systems (Buchberger et al., 2010).
1.2.2.2. The SREBP signalling processes of the ER and Golgi.
The control of cholesterol levels in human cells is mediated by the Sterol Receptor Element Binding Protein Pathway (SREBP). When cholesterol levels are low, SREBP is released from the endomembrane system to activate gene involved in cholesterol biosynthesis, of which there are about 30 in humans (Espenshade and Hughes, 2007). In humans, there are two SREBP genes that encode three proteins. Each protein controls the expression of genes to make a particular class of fatty acid or cholesterol. Under conditions of high cholesterol, SREPBs are held in the ER, but under conditions of low fatty acid/cholesterol, the SREBPs are released to migrate to the Golgi. In the Golgi, the two Site proteases Site-1 and Site-2 cleave SREBP releasing the N-terminal portion to enter into the nucleus and activate the transcription of target genes (Todd et al., 2006). The SREBP system is conserved in S. pombe, where it regulates the synthesis of the sterol ergosterol (Hughes et al., 2005). Interestingly, it is also the main system for low oxygen sensing and the regulation of hypoxia induced gene expression (Todd et al., 2005). In both humans and S. pombe, the cleavage of the N-terminus of SREBP (Sre1 in S. pombe) is initiated by their ubiquitylation by Golgi-resident E3 ubiquitin ligases (Stewart et al., 2011).
1.3 Human diseases associated with Golgi malfunction
The basic function of the Golgi complex is to locate and transport of proteins within and out of the cell. It receives proteins and lipids through the cis-face, they are modified as they traverse the Golgi and they exit through the trans-face. The failure for this process to work correctly has been associated with a number of human diseases. Alzheimer’s disease is a brain disorder due to the accumulation of Beta-amyloid plaques in brain cells. It has been observed that disruption of Golgi structure comes before the accumulation of protein plaques (Joshi et al., 2014). They report that Aβ signals Golgi fragmentation, which in turn increases APP trafficking leading to accelerated Aβ production in a feed-forward loop. The amyloid-beta (Aβ) also raises the levels of cell cycle kinase, Cdk5, which phosphorylates GM130 leading to Golgi dispersal (Sun et al., 2008). Therefore, they concluded that there are two ways to reverse or prevent the harmful effects of beta-amyloid secretion in AD patients, first by inhibiting or a mutated Cdk5 or degrading increasing APP protein degradation through the ubiquitin-proteosome pathway. The disruption of Golgi structure has also been implicated in other neurological disorders, such as Parkinson’s and Creutzfeldt-Jakob diseases (Gonatas et al., 2006).
The previous example demonstrated how signalling processes can disrupt Golgi function. There are many examples where altered function of a Golgi protein itself results in a severe disease (Bexiga and Simpson, 2013). This would be expected for a process deciding the localisation of so many numbers of proteins. In addition to that, there are many ways by which the Golgi can be affected. For example, a defect in retrograde protein transport may have as severe effects as a disruption to anterograde transport. Over the past few years, many diseases have been linked to the malfunction of the Golgi complex, including skin diseases, neurological disorders, skeletal dysplasia and connective tissue disorders. I will briefly mention some of them here. An example of altered protein trafficking for a skin disorder is Dyschromatosis universalis hereditaria, which commonly occurs in Japan (Zhang et al., 2013a). A defect in the gene ABCB6 gene leads to accumulation of its translated protein in the Golgi causing malfunction. An example of neurological disease comes from a defect in the myelin gene, PLP1. A mutation in
PLP1 causes a rare disease known as Pelizaeus-Merzbacher syndrome. This disease results
from an accumulation of the misfolded PLP1 protein remaining in the ER resulting in a defect in retrograde transport of proteins from the Golgi to the ER, contributing to Golgi fragmentation (Inoue, 2005). Another Neurological disease proximal spinal muscular atrophy is caused by low expression of the SMN1 gene leading to accumulation of SMN granules in the late stage of Golgi network and stopping vesicle transport (Ting et al., 2012). Also, North Sea myoclonus neurological disease is where a mutant cannot localise the protein to the cis-Golgi. This has
been linked to the X-linked mental retardation 1 (MRX1) and is caused by mutation in the
IQSEC2 gene located on chromosome Xp11.2 (Boissé Lomax et al., 2013; Corbett et al., 2011).
Many malfunctioning proteins found to cause diseases have been classified under connective tissue disorders. In these cases, disrupted traffic through the Golgi does not all proper delivery of connective tissue proteins to the extracellular matrix. Examples of these are RIN2 syndrome,
MACS syndrome, Gerodermia osteodysplastica, Menkes disease and Cutis laxa (Bexiga and
Simpson, 2013). One final example for a possible link of skeletal diseases to Golgi function is through the uncharacterized transmembrane protein TMEM165 (Foulquier et al., 2012). The wild-type protein is in the Golgi complex and to the endosomal-lysosomal system. In human cells lacking this protein, trafficked protein are spread to different locations around the cell. It appears that TMEM165 has important role in Golgi glycosylation and Golgi morphological structure.
1.3.1 Skeletal dysplasia
There are two causes of the skeletal dysplasia Dyggve-Melchior-Clausen syndrome (DMC) that I will discuss. The first is caused by mutations that alter or eliminate the function of the RAB33B and the second is the protein DYMECLIN (DYM). DMC syndrome is characterized by short stature due to skeletal dysplasia deformities accompanied by microcephaly and intellectual delay (Dyggve et al., 1962). DMC is very similar to another genetic syndrome called Smith-McCort dysplasia (SMC). Smith-Smith-McCort dysplasia is distinguished from DMC by the lack of mental retardation, but the skeletal deformities are similar (Khalifa et al., 2011). Both DMC and SMC are autosomal recessive disorders in that in the disease occurs when a child inherits mutant alleles from each parent and so is homozygous for mutations at that locus. Heterozygous individuals do not appear to be affected by the disease.
parent and so is homozygous for mutations at that locus. Heterozygous individuals do not appear to be affected by the disease.
This syndrome has been reported in many countries around the world and early diagnosis is important. Preventing the disease by counselling parents to avoid passing the disease on to children is the only current means of preventing the disease, since it is a standard recessive genetic disease (Bayrak et al., 2005; Khalifa et al., 2011). Early treatment is important to alleviate some of the aggravation of the disease, such as using surgery to join vertebrae called spinal fusion or other surgical techniques to correct obvious skeletal abnormalities. Nowadays, pre-implantation genetic diagnosis can be used for those parents who appear to be at risk to pass the syndrome to their children. Although it is rare syndrome, it appears that the
number of cases continues to increase. In 2002 there were only 58 cases known and this increased to 90 cases in 2004 (Kandziora et al., 2002) with 15 cases attributed to nine unrelated families in Egypt with both males and females being affected by a defective DYM gene (Santos et al., 2009).
1.3.1.1 Malfunctioning RAB33B as a cause of SmithMcCort dysplasia
RAB33B was the second locus found where mutation led to the DMC syndrome (Alshammari et al., 2012). RAB33B resides on chromosome 4 at position 4q31.1 and homozygosity of mutation leading to complete malfunction of the protein is the cause of DMC. There are around 70 different RAB proteins in the human genome and they have been found in a variety of organelles (Bem et al., 2011; Liegel et al., 2013). RAB proteins are small intracellular GTPase involved in cellular protein trafficking. Seven of them are involved in Golgi structural maintenance, Rab1, Rab2, Rab6, Rab18, Rab33B, and Rab43. These RAB proteins likely serve to recruit other factors to Golgi membrane. Like other GTPases, such as the Ras and Rho GTPases, these cycle between a GTP-bound and a GDP-bound form. In the GTP bound form they are attached to the Golgi membrane to carry out their recruitment function. Rab GTPases have low GTPase activity, but are activated by guanine nucleotide exchange factors (GEFs) (Martínez-Alonso et al., 2013), which is likely their mechanism of regulation. The Rab proteins are believed to be the link between Golgi function and the regulation of protein transport (Liu and Storrie, 2012) and the identification of loss of RAB33B leading to DMC is strongly supports this. RAB33B is specifically thought to be involved in retrograde protein transport through the Golgi, which also shows that anterograde and retrograde movement are closely linked (Dupuis et al., 2015). Several other human diseases are caused by RAB mutations including cancer. For example RAB18, RAB1GAP and RAB2GAP have been implicated Warburg Micro syndrome (Bem et al., 2011; Liegel et al., 2013) and VPS53 in progressive cerebello-cerebral atrophy type 2 (Feinstein et al., 2014).
1.3.1.2 Lack of DYMECLIN function resulting in DMC syndrome.
The Dymeclin (DYM) gene responsible for DMC maps to chromosome position 18q12-21.1 (El Ghouzzi et al., 2003). The name DYMECLIN comes from the combination of the researchers from Denmark that first systematically reported the disease Dyggve, Melchior and Clausen (Dyggve et al., 1962). This gene encodes a peripheral membrane protein located in the Golgi (Dimitrov et al., 2009) that is involved in protein trafficking (Osipovich et al., 2008). The DYM protein appears to be able to shuttle back and forth between the cytosol and Golgi, thus it
is likely tethered to the Golgi by fatty acid modification of the protein (Denais et al., 2011; Dimitrov et al., 2009). The cases of DMC that have been shown to involve DYMECLIN result from mutations in the gene that eliminate the protein (Khalifa et al., 2011). In cells of DYM patients there were ultrastructural analomolies to organelles within cells, but the Golgi apparatus appeared normal (El Ghouzzi et al., 2003).
1.4 Cancer as a genetic disease
The human body contains a huge number of cells and every cell, with the notable exceptions of red blood cells and platelets, contain a nucleus, where the majority of them contain 23 pairs of chromosomes, one of each inherited from the mother and father. As mentioned above for the cases of heritable genetic diseases, a pre-disposition to cancer can also be passed from parents to children. Every cell in our body has about 20,000 to 25,000 genes, of which there have been 20203 annotated proteins (www.expasy.org). This is likely to be a large underestimate of the true number of functional genes if non-coding RNAs are considered (Griffiths-Jones, 2007). In any cell type, around half of these are activate to make a certain cell type and of this half, two different cell types differ in protein-coding genes expressed by about 2000. Thus each cell probably is different by about 2000 proteins.
Cancer is a disease when the gene expression program in cells gets out of whack. Cancer can start from the gene expression mis-programming of one cell and the replication of this cell could lead to abnormal growth of cells in a group called a tumour. Once started, it can spread to other parts, called metastasis. Thus, the healthy tissues will be affected by these cells with over time either by the tumour growing and changing the local tissue environment or spreading to new tissues (Bertram, 2001). There are about 15 million new cases of cancer each year (Ferlay et al., 2014). Approximately, there are 200 types of cancers, and based on that it is classified as heterogeneous disease which may causes by different kind of factors. The most common types of cancers are breast, lung, colorectal and prostate (Ferlay et al., 2014). Many studies indicate that cancer caused by both environmental and genetic (heredity) factors. Many genetic mutations do not affect cells and cells have the ability to repair the majority of them. Not all mutations have the same effective as some prevent proteins from being made, and others alter the function of the protein made, or a mutation causes more protein made than required. Genetic mutations can occur by different ways, such as point mutations, deletions, and insertions. Point mutations are the most common, where one base is changed to another. The effect of a mutation is also dependent on the type of cell in which the mutation occurs, for example, reproductive cells going through meiosis or somatic cells undergoing mitosis (Wilkins
